Effect of Additives on Refolding of a Denatured Protein

Masahiro Yasuda,* Yumi Murakami, Ayumu Sowa, Hiroyasu Ogino, andHaruo Ishikawa

Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531,Japan

Denatured lysozyme was refolded by a dilution method. The refolding yield dependedgreatly on the lysozyme concentration in the refolding mixture. When the concentra-tion of denatured lysozyme was 0.02 g/L, the refolding yield was as high as 60%.However, when the concentration of denatured lysozyme was 0.2 g/L, the refoldingyield was as low as 10% due to the formation of aggregates. To prevent the formationof aggregates and to increase the refolding yield at a low cost, inexpensive additiveswere screened. The addition of acetone, acetoamide, or urea derivatives was veryeffective for improving the refolding yield. To clarify why the addition of acetoamidein the refolding mixture improved the refolding yield at the high lysozyme concentra-tion, the time courses of the concentration and the average diameter of the aggregatesin the refolding mixture were monitored by the dynamic light scattering method. Theexperimental results showed that acetoamide played a role in preventing the formationand growth of aggregates and secondary aggregation between the lysozyme aggregates.

IntroductionRecently, several commercially important proteins

have begun to be produced by the recombinant DNAtechnology. Escherichia coli is often used as a host cellof the recombinant DNA because of its rapid growth andgreat rate of protein production. However, when E. coliis used as a host cell, the overexpressed protein usuallyresults in formation of inclusion bodies (Prouty andGoldberg, 1972; Prouty et al., 1975). Although theinclusion bodies can easily be recovered from the cellsby disruption, their proteins are in aggregated, concen-trated, and denatured forms. To obtain native proteinsfrom the inclusion bodies, it is necessary to dissolve themin a buffer containing a denaturant, such as guanidinehydrochloride (GuHCl), urea, or surfactant, followed byoxidation of the thiol groups, if necessary (Ikatura et al.,1977), and to submit the solubilized protein to a refoldingprocedure (Ikatura et al., 1977; Rudolph, 1990). Therefolding yield of the denatured proteins depends on thespecific amino acid sequence of the protein and on therefolding condition. From an industrial viewpoint, re-folding should be performed at high protein concentra-tions. Reduction of the volume of solutions to be pro-cessed would reduce the processing time and cost.However, it has been observed that the refolding yielddecreases with an increase in the concentration of theprotein to be refolded (Goldberg et al., 1991; Jaenicke,1974; London et al., 1974; Teipel and Koshland, 1971;Wetlaufer et al., 1974). When refolding was performedat high protein concentrations, denatured aggregateswere the predominant products.

It has been shown that two types of interactions, thatis, intermolecular and intramolecular interactions, oc-curred competitively during the refolding process andthat the former interaction was responsible for thedecreased refolding yield as a function of the protein

concentration (Goldberg et al., 1991; Zettlmeissl et al.,1979). The intramolecular interaction is essentiallyunimolecular, and therefore, the rate of formation ofrenatured protein should not depend on the proteinconcentration. The intermolecular interaction corre-sponds to a multimolecular reaction; consequently, therate of formation of aggregates should increase rapidlywith the protein concentration. At lower protein con-centrations, aggregation should occur slowly, and theintramolecular interaction leading to the native formwould be expected to prevail over aggregation. At highprotein concentrations, the aggregation process shouldbe faster than the first-order intramolecular interaction,resulting in a reduced refolding yield.

Industrially, formation of the protein aggregates is aserious problem because it reduces the refolding yield.There have been several attempts to prevent the forma-tion of the protein aggregates. Stepwise dilution of thedenaturant improved the refolding yield of reducedchymotrypsinogen A in a batch reactor (Orsini andGoldberg, 1978; Orsini et al., 1975) and lysozyme in aflow-type reactor (Terashima et al., 1996).

On the basis of the idea that individual proteinmolecules would be refolded correctly when they wereisolated from one another during refolding, RNase wasrefolded in a reversed micellar system (Hagen et al.,1990). In this system, protein aggregation was fullyprevented. However, the GuHCl concentration must bereduced to as low as possible to enhance transfer ofdenatured protein to reversed micelles. A low GuHClconcentration will result in protein aggregation beforethe protein molecules are entrapped into the reversemicelles. This disadvantage was partly overcome byoptimizing the reversed micellar system (Garza-Ramoset al., 1992).

Recently, some additives have been shown to beeffective in assisting protein folding (Zardenta andHorowitz, 1994a). Such additives were low concentra-* Telephone: +81-722-54-9299. FAX: +81-722-54-9911.

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tions of detergents (Wetlaufer and Xie, 1995), liposome(Zardenta and Horowitz, 1994b), poly(ethylene glycol)(Cleland et al., 1992), and cyclodextrins (Karuppiah andSharma, 1995). These additives promote refolding of theproteins by binding critical interactive sites of denaturedproteins, thereby preventing misfolding and/or aggrega-tion.

In addition to the methods described above, there havebeen several other investigations concerning the refoldingof denatured proteins. However, from the viewpoint ofindustrial application, previous methods are not neces-sarily efficient because of the complexity and cost of therefolding process.

Therefore, to develop a simple and inexpensive refold-ing process, we studied a dilution method which usednonproteinaceous additives. Lysozyme was used as amodel protein and the denatured lysozyme was refoldedby the dilution method in the presence of various addi-tives. The effects of the additives on the refolding yieldand the formation of aggregates were investigated.

Materials and Methods

Chemicals and Enzyme. All the reagents were ofthe highest grade available commercially and were usedwithout further purification. The following additiveswere obtained accordingly: Polyoxyethylene nonylphenylether (Triton N-57) was purchased from Sigma ChemicalCo. Ltd. (St. Louis, MO), tricaprylylmethylammoniumchloride (Aliquat 336) from Aldrich Chemical Co. Inc.(Milwaukee, WI), and urea from Wako Pure ChemicalsCo. Ltd. (Osaka, Japan). All the other additives andGuHCl, 2-mercaptoethanol, and glutathione of oxidizedform and reduced form were purchased from NacaraiTesque (Kyoto, Japan). Hen egg white lysozyme andMicrococcus lysodekticus dried cells were purchased fromWako.

Preparation of Denatured Lysozyme. Weighedlysozyme was dissolved in 0.1 M Tris-HCl buffer (pH 8.5)containing 1 mM EDTA (TE buffer) and a denaturantand a reductant of various concentrations. GuHCl wasused as a denaturant and 2-mercaptoethanol as a reduc-tant. The final concentrations of lysozyme, the denatur-ant, and the reductant were 1.2-12 g/L, 6 M, and 154µM, respectively. The mixtures were bubbled with dryN2 for 10 min and incubated for 120 min at 40 °C todenature the lysozyme completely. The mixtures werestored as a stock solution of the denatured lysozyme andused for all the refolding experiments.

Refolding of Denatured Lysozyme. Refolding ofthe denatured lysozyme was performed by diluting thestock solution containing 1.2-12 g/L denatured lysozymewith 0.1 M buffer solution. The total volume of the eachrefolding mixture was 0.5 mL. This mixture was firstmixed by using a vibrating mixer for about 2 s and thenplaced in a water bath kept at 30-50 °C without shaking.Prior to the experiments concerning the effect of thelysozyme and GuHCl concentrations and the addition ofvarious additives on the refolding yield, optimal refoldingconditions were studied at a low lysozyme concentrationof 0.02 g/L. Under the above optimal refolding conditions,except for the GuHCl concentration, the denaturedlysozyme was refolded mainly at a high lysozyme con-centration of 0.2 g/L to study the effect of the GuHClconcentration and the addition of additives on the refold-ing yield.

Enzyme Assays. The lysozyme activity was deter-mined at 30 °C by following the decrease in absorbanceat 450 nm of a 0.50 g/L suspension of M. lysodekticus

dried cells in 0.06 M potassium phosphate (pH 6.2). Theassay volume was 1 mL. The refolding yield wasdetermined as the ratio of the activity recovered afterrefolding to that of the native lysozyme before denatur-ation.

Measurement of the Average Diameter and theConcentration of Aggregated Proteins. The averagediameter and the concentration of the aggregated pro-teins were measured by the dynamic light scatteringmethod using an Otuka DLS-700 dynamic light scatter-ing photometer (Otuka Electronics, Osaka, Japan)equipped with a 5 mW He-Ne laser. Refolding wasperformed in a measuring glass cell of the dynamic lightscattering photometer, which was incubated at 35 °C.

Results and DiscussionEffect of Lysozyme Concentration on Refolding.

Lysozyme is one of the enzymes that are most extensivelystudied for their refolding properties. The denatured andreduced lysozyme was refolded in the presence of amixture of oxidized form and reduced form glutathiones.In the present experiments, refolding was performed bydiluting the stock solution containing the denaturedlysozyme with the refolding buffer to predeterminedprotein concentrations.

Prior to performing the experiments concerning theeffect of addition of various additives on the refoldingyield, the effects of refolding conditions on the refoldingyield were studied. Table 1 summarizes the experimen-tal results of the optimal refolding conditions at a lowlysozyme concentration of 0.02 g/L. These results weresimilar to those obtained by other investigators (Wet-laufer et al., 1974; Goldberg et al., 1991).

Next we studied the effect of the lysozyme concentra-tion on the time course of refolding. The denaturedlysozyme was refolded under the optimal conditionsshown in Table 1. Figure 1 shows the time courses ofrefolding in which the refolding yields, measured underthe three lysozyme concentrations, increased with timeand attained equilibrium values at about 30 min. Asshown in the figure, the refolding yield decreases withthe increase in the lysozyme concentration.

It was anticipated that the refolding yields at thehigher lysozyme concentrations would improve by opti-mizing the GuHCl concentration. Therefore, the effectof the GuHCl concentration on refolding of 0.2 g/Ldenatured lysozyme was investigated. The refoldingconditions except for the GuHCl concentration were thesame as those shown in Table 1. In Figure 2, the closedcircles (b) show the experimental results observed at 120min incubation time. The other data (O and 4) will bediscussed later. The refolding yield improved to as highas about 60% at the optimal GuHCl concentration ofabout 1.5 M. When the GuHCl concentrations werelower than 1 M, formation of the lysozyme aggregateswas observed immediately after dilution of the enzymestock solution with the refolding buffer. Thus, thedecrease in the refolding yield at the higher lysozyme

Table 1. Optimal Refolding Conditions at a LowLysozyme Concentration of 0.02 g/L

refolding temp 35 °CpH of refolding mixture 8.5components of refolding mixture 0.1 M Tris-HCl

1 mM EDTA5 mM oxidized form of

glutathione (GSSG)5 mM reduced form of

glutathione (GSH)0.1 M GuHCl

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concentrations had to be strongly related to the formationof aggregates. When the GuHCl concentration washigher than 1.5 M, the refolding yield decreased with theincrease in the GuHCl concentration. This is becauseGuHCl, the denaturant, denatured the refolded lysozymeand/or prevented refolding of the denatured lysozyme.

Effect of Additives on Lysozyme Refolding at aHigh Lysozyme Concentration. Refolding of thedenatured protein at higher concentrations is desirableindustrially because it can reduce the volume of thesolutions to be handled, the processing time, and cost.As shown above, considerable improvement in the refold-ing yield at a higher protein concentration was possibleby optimizing the GuHCl concentration. However, it isnot necessarily the best for the industrial applicationbecause a large amount of expensive GuHCl must beused.

Therefore, we screened inexpensive additives whichcould prevent the formation of aggregates and increasethe refolding yield at a low cost. We first examined awater-soluble polymer, surfactants, acetone, acetoamide,urea, trimethylamine hydrochloride, and dimethyl sul-foxide. Figure 3 compares the experimental refoldingyields attained at 120 min incubation time in the pres-ence of various additives with that observed with noadditives. In the figure, the relative refolding yieldobserved with no additives was taken as 1.0. The GuHCl

concentration was maintained at as low as 0.1 M. Theadditive concentration was 1.4 M except for the surfac-tants and the water-soluble polymers. The concentra-tions of Tritons and Aliquat were all 10 mM and thoseof the water-soluble polymers were 0.05% (w/v). Themolecular weight of poly(vinylpyrrolidone) (PVP) wasabout 30 000 and that of poly(ethylene glycol) (PEG) was20 000. When the refolding buffer contained no addi-tives, the refolding yield was only about 12%. As can beseen in Figure 3, the addition of acetone, acetoamide,urea, or dimethyl sulfoxide is very effective for improvingthe refolding yields. These chemicals have molecularstructures similar to GuHCl. GuHCl has a carbon-nitrogen double bond and two amino groups as functionalgroups. Acetoamide, acetone, and dimethyl sulfoxidehave a carbon-oxygen double bond (carbonyl group) ora sulfur-oxygen double bond (sulfonyl group), both ofwhich are similar to the carbon-nitrogen double bond.Due to the improvement of the refolding yield, thesefunctional groups seemed to play an important role inlysozyme refolding at a high lysozyme concentration. Onthe other hand, the water-soluble polymers PVP and PEGand the surfactants had almost no effect on the refoldingyield of lysozyme. Goldberg et al. (1991) also found thatthe addition of PEG was not effective for improving therefolding yield of lysozyme. However, they found thatthe addition of PEG was effective for improving therefolding yield of carbonic anhydrase B. These resultsmean that the effect of the water-soluble polymers on therefolding yield depends on the enzymes.

In the above experiments, the GuHCl concentrationwas maintained at as low as 0.1 M. The refolding yieldsin the presence of additives were also expected to dependon the GuHCl concentration. Therefore, the effect of theGuHCl concentration on refolding in the presence of 1.4M acetone and 1.4 M acetoamide was investigated. Therefolding conditions, except for the GuHCl concentration,were the same as those shown in Table 1. The experi-mental results observed at 120 min incubation time werecompared with those obtained in the absence of theadditive in Figure 2. As mentioned above, when therewere no additives in the refolding mixture, the optimalGuHCl concentration was about 1.5 M. However, whenthe refolding mixture contained the additives, the optimalGuHCl concentration decreased to 0.7 M because addi-

Figure 1. Effect of the lysozyme concentration on refolding.Denatured lysozyme was refolded under the optimal conditionsshown in Table 1 except for the lysozyme concentration. Thefinal lysozyme concentrations were as follows: 0.02 (O), 0.067(4), and 0.2 g/L (0).

Figure 2. Effect of the GuHCl concentration on the refoldingyield. Denatured lysozyme was refolded under the optimalconditions shown in Table 1 except for the GuHCl concentration.The lysozyme concentration was 0.2 g/L. The GuHCl concentra-tion was varied between 0.1 and 4 M. The additive concentra-tions were 1.4 M. The refolding yield was measured after 120min incubation.

Figure 3. Lysozyme refolding in the presence of variousadditives. Denatured lysozyme was refolded under the optimalconditions shown in Table 1. The final lysozyme concentrationwas 0.2 g/L. The additive concentration was 1.4 M except forsurfactants and water-soluble polymers. Tritons and Aliquatconcentrations were 10 mM. PVP and PEG concentrations were0.05% (w/v). The refolding yield was measured after 120 minincubation.

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tives such as acetoamide and acetone also played animportant role in preventing the lysozyme aggregation.Furthermore, when the refolding mixture contained 1.4M acetoamide or acetone, the refolding yield improvedconsiderably. However, acetoamide is a more efficientadditive than acetone for improving the refolding yield.

As can be seen in Figure 2, GuHCl not only preventsprotein aggregation but also interferes with proteinrefolding. However, acetoamide prevents protein ag-gregation without interfering with protein refolding. Infact, when 3 M acetoamide was added to the refoldingmixture, the refolding yield was the same as thatobtained in the presence of 1.4 M acetoamide (data notshown).

As shown in Figure 3, additives whose molecularstructures are similar to GuHCl improve the refoldingyield at a high lysozyme concentration. To investigatewhat kinds of functional groups of the additives wereeffective for improving the refolding yield, we furtherexamined various additives under the condition of 0.7 MGuHCl. We chose urea derivatives, ketones, amides, analdehyde, an ester, and surfactants as the additives toimprove the refolding yield. The denatured lysozyme wasrefolded under the optimal conditions shown in Table 1,except for the GuHCl concentration. The additive con-centration was 1.4 M, except for the surfactants. SodiumN-lauroylsarcosinate and sodium dodecyl sulfate concen-trations were 0.05% (w/v). Aerosol OT and Tweensconcentrations were 10 mM. Figure 4 compares therelative refolding yields observed in the presence ofvarious additives with one observed with no additives.

The refolding yield observed with no additives was about50%, and its relative refolding yield was taken as 1.0.As shown in the figure, the urea derivatives and amidesimproved the refolding yield while the aldehyde, ester,and surfactants tested decreased the refolding yield.Although acetoamide was the best additive when themixture contained 0.1 M GuHCl, as shown in Figure 3,the urea derivatives were the most effective additiveswhen the mixture contained 0.7 M GuHCl. This may beattributed to the change in the interaction between theadditive and the refolding intermediates with the GuHClconcentration.

Effect of Acetoamide on the Lysozyme Aggrega-tion. As shown above, the refolding yield at the highlysozyme concentration improved considerably whenadditives such as acetoamide were added to the refoldingmixture. Next, we studied the effect of the additives onthe lysozyme aggregation. As shown in Figure 2, whenthe GuHCl concentration was 0.1 M, the refolding yieldwas as low as 10%. This low refolding yield was at-tributed to the formation of the lysozyme aggregateswhich was observed immediately after dilution. Whenacetoamide was added under the same conditions exceptfor the existence of acetoamide, the formation of thelysozyme aggregates was lower in comparison with thecase of no addition. To investigate quantitatively theeffect of the additive on formation of aggregates, wemonitored the time courses of the concentration and theaverage diameter of the aggregates in the refoldingmixture by the dynamic light scattering method. Whenthe final GuHCl concentration was 0.1 M, the average

Figure 4. Effect of additives on the relative refolding yield at a GuHCl concentration of 0.7 M. Denatured lysozyme was refoldedunder the optimal conditions shown in Table 1 except for the GuHCl concentration. The lysozyme concentration was 0.2 g/L. TheGuHCl concentration was 0.7 M. The additive concentration was 1.4 M except for surfactants. Sodium N-lauroylsarcosinate andsodium dodecyl sulfate concentrations were 0.05% (w/v). Aerosol OT and Tweens concentrations were 10 mM. The refolding yieldwas measured after 120 min incubation.

604 Biotechnol. Prog., 1998, Vol. 14, No. 4

diameter of the protein aggregates increased too fast tobe monitored by the dynamic light scattering photometerwhether the refolding mixture contained 1.4 M aceto-amide or not. Therefore, the final GuHCl concentrationwas increased up to 0.7 M to decrease the rate of growthof the protein aggregates. Other refolding conditionswere the same as those shown in Table 1.

Figure 5 shows the experimental time courses of theconcentration of the aggregates. The concentration of theaggregates decreased rapidly with the incubation timewhen acetoamide was not added, indicating that asecondary aggregation between the lysozyme aggregatestook place. On the other hand, the concentration of theaggregates increased very slowly with time when aceto-amide was added. This means that acetoamide is veryeffective for preventing the formation of aggregatesconsisting of unfolded or misfolded lysozyme and second-ary aggregation between the lysozyme aggregates.

Figure 6 shows the effect of the acetoamide additionon the time courses of the average diameter of thelysozyme aggregates. The average diameter of the ag-gregates increased rapidly with the incubation time whenacetoamide was not added. However, the average diam-eter of the aggregates increased slowly with the incuba-tion time when acetoamide was added. The aggregatesseemed to grow by absorbing the aggregates and mis-folded or unfolded protein.

From the results shown in Figures 5 and 6, it wasconcluded that acetoamide played an important role inpreventing the formation and growth of the lysozymeaggregates and secondary aggregation between them.Other additives which were found in the present workto improve the refolding yield at the high lysozymeconcentration were also thought to prevent the formationand growth of its aggregates.

ConclusionsThe present work was undertaken to develop an

efficient refolding process of protein. Lysozyme was usedas a model protein. The refolding yield measured underthe optimal conditions shown in Table 1 depended greatlyon the lysozyme concentration in the refolding mixture.When 0.2 g/L denatured lysozyme was refolded under theabove optimal conditions, the refolding yield was as lowas 10% due to the formation of lysozyme aggregates.When the GuHCl concentration in the refolding mixturewas optimized (the optimal GuHCl concentration wasfound to be 1.5 M), the refolding yield improved to as highas about 60%. However, this condition is not necessarily

the best for the industrial application because a largeamount of expensive GuHCl must be used.

To prevent the formation of the aggregates and toincrease the refolding yield at a low cost, inexpensiveadditives were screened. Water-soluble polymers, sur-factants, and some chemicals of which the molecularstructures are similar to GuHCl were examined. Thealdehyde, ester, water-soluble polymers, and surfactantsdecreased the refolding yield. However, when the refold-ing mixture contained acetoamide, acetone, urea, anddimethyl sulfoxide, the refolding yield improved consider-ably.

To investigate quantitatively the effect of acetoamideon formation of aggregates, we monitored the timecourses of the concentration and the average diameterof the aggregates in the refolding mixture by the dynamiclight scattering method. From this experiment, it wasconcluded that acetoamide played an important role inpreventing the formation and growth of the lysozymeaggregates and secondary aggregation between them.

We demonstrated that the addition of the additives,such as acetoamide and acetone, improved the refoldingyield at the high lysozyme concentration. This wouldreduce the volume of solutions to be handled and theprocessing time. Both acetoamide and acetone areinexpensive and commercially available and are easilyseparated from solutions containing the refolded proteinby an appropriate separation method, such as dialysis.Furthermore, refolding in the presence of additives doesnot require special and/or additional equipment. There-fore, the refolding in the presence of additives such asacetoamide should be applicable industrially.

Acknowledgment

This work was (partly) supported by the SasagawaScientific Research Grant from The Japan Science Soci-ety.

References and Notes

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Figure 5. Time courses of the concentration of protein ag-gregates. Denatured lysozyme was refolded under the optimalconditions shown in Table 1 except for the GuHCl concentration.The lysozyme concentration was 0.2 g/L. The GuHCl concentra-tion was 0.7 M. The acetoamide concentration was 1.4 M.

Figure 6. Time courses of the average diameter of proteinaggregates. Denatured lysozyme was refolded under the optimalconditions shown in Table 1 except for the GuHCl concentration.The lysozyme concentration was 0.2 g/L. The GuHCl concentra-tion was 0.7 M. The acetoamide concentration was 1.4 M.

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Accepted May 15, 1998.

BP9800438

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